Project Summary: Intracellular Ca2+ signaling via changes in cytosolic Ca2+ concentration controls a wide range of cellular and physiologic processes. Ca2+ mobilization from intracellular stores mediated by second messengers plays a critical role in regulation of cytosolic Ca2+ levels. Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+-mobilizing second messenger identified to date; it uniquely mobilizes Ca2+ from acidic endolysosomal organelles. NAADP has been shown to be effective in evoking Ca2+ release in a multitude of different mammalian cells and defects in NAADP signaling are now being implicated in many diseases. Despite the importance of NAADP-evoked Ca2+ signaling, the molecular identity of the NAADP receptor remains elusive and the Ca2+-release channels involved in this process have yet to be unequivocally defined. Accumulated evidence indicates that endolysosomal two-pore channels (TPCs) play an important role in NAADP-evoked Ca2+ release. However, strong evidence also suggests that TPCs are not NAADP receptors. Currently, a unifying hypothesis is that TPCs are the key channels responsible for NAADP-evoked Ca2+ release but that their NAADP sensitivity arises from some unknown NAADP-binding accessory protein. To identify the elusive NAADP receptor, we used both TPCs and NAADP as bait to isolate their interacting partners from mammalian HEK293 and SKBR3 cells and then used an unbiased quantitative proteomic analysis and a Ca2+- imaging functional assay to screen and identify novel proteins that are important for NAADP-evoked Ca2+ release. We have identified an Lsm12 protein to be an interacting protein of both NAADP and TPCs. With Lsm12-knockout cells, we found that Lsm12 mediates the interaction between NAADP and TPCs and is essentially required for NAADP-evoked Ca2+ release in HEK293 cells. We hypothesize that Lsm12 is essential and directly involved in NAADP-evoked Ca2+ release by functioning as a NAADP receptor and/or a TPC regulatory or auxiliary protein. We will pursue the following 3 specific aims: 1) test the hypothesis that Lsm12 is essential and directly involved in NAADP-evoked ca2+ release; 2) test the hypothesis that Lsm12 is a NAADP receptor; and 3) test the hypothesis that Lsm12 is a regulatory or auxiliary protein of TPCs. We will achieve these goals by using multidisciplinary approaches from molecular biology, cell biology, protein biochemistry, and electrophysiology. Findings from the proposed research will provide a breakthrough that will advance our understanding of the molecular basis and mechanisms of NAADP-evoked Ca2+ release, and facilitate the development of new drugs for this important Ca2+ signaling process.